The invention relates to methods and instruments for measuring daughter-ion spectra (also known as fragment-ion spectra or MS/MS spectra) in time-of-flight mass spectrometers, especially those with reflectors, with post-acceleration of selected parent and daughter ions by raising the potential of a xe2x80x9cpotential lift cellxe2x80x9d during the passage of the ions.
In a time-of-flight mass spectrometer, the mass-to-charge ratio m/z of ions can be determined from their time of flight. Although it is always the mass-to-charge ratio m/z which is measured in mass spectrometry, with m being the mass and z being the number of elemental charges carried by the ion, in the following, for the sake of simplicity, only the mass m and its determination will be referred to. Since many types of ionization, such as MALDI, predominantly supply only single-charged ions (z=1), the difference ceases to exist in practice for these types of ionization.
In a time-of-flight mass spectrometer which is equipped with an ion selector and a velocity-focusing reflector, it is possible to measure the daughter-ion or fragment-ion spectra of parent ions which are selected by the ion selector on the basis of their time of flight. The decay of parent ions into daughter or fragment ions can be induced by introducing excess energy during ionization (so-called PSD xe2x80x9cPost Source Decayxe2x80x9d spectra) or by applying other methods such as collisionally induced fragmentation (so-called CID xe2x80x9cCollisionally Induced Decompositionxe2x80x9d spectra).
The two-stage ion reflector according to Mamyrin has achieved considerable popularity as a velocity-focusing reflector. The ions are strongly decelerated during the initial brake stage of the reflector but only weakly decelerated in the second deceleration stage. The faster ions penetrate further than the slower ions into the linear, relatively weak deceleration field of the second deceleration stage of the reflector and therefore travel for a greater distance. With proper adjustment of the two deceleration fields, this difference in distances can be used to compensate for the faster time-of-flight velocity of the ions from a primary focus so that they arrive at the secondary focus at precisely the same time. The focal length of the velocity-focusing device is slightly energy dependent.
The parent ions and the daughter ions resulting from their decay enter the reflector simultaneously with the same average velocity but with different mass-proportional energies, such that they will be dispersed according to their mass within the reflector by their different energies. However, this method of detecting daughter or fragment ions by using these types of reflectors has serious disadvantages. With reasonably good focusing, only ions within a relatively small energy range can be detectedxe2x80x94in the commercially available instruments of standard design, this represents approximately 25-30% of the energy range. The reason for this is that the ions always have to pass through the first deceleration field in order to achieve velocity-focused reflection. However, the first deceleration field consumes a good ⅔ of the original acceleration energy. This means that, from parent ions with an initial mass of 3200 atomic mass units, only those fragments between about 2400 and 3200 atomic mass units can be scanned in an initial fragment-ion segment spectrum; only those between 1800 and 2400 mass units can be scanned in a second segment spectrum with reduced reflector voltage, and only those between 1350 and 1800 can be scanned in a third segment spectrum etc. Thus, for an average sized peptide, approximately 10 to 15 segment spectra have to be scanned in order to measure the whole fragment-ion spectrum. Then, a complicated mass-calibration procedure has to be applied to get all the masses from the segment spectra. Only after all these segment spectra have been pasted together, can the daughter ion spectrum be evaluated in the data system as an artificially generated single composite spectrum.
According to the patent application GB 2 344 454 (German patent DE 198 56 014), methods have now been put forward for recording daughter-ion spectra in a single scan using either a linear time-of-flight mass spectrometer, or a time-of-flight mass spectrometer equipped with a two-stage ion reflector. The patent application also describes PSD, CID, MALDI (Matrix Assisted Laser Desorption and Ionization) and velocity focusing by delayed acceleration in the ion source.
One of the proposed methods consists of subjecting the ions to relatively mild acceleration in the ion source (using an acceleration of the ions which is slightly delayed with respect to the ion-producing laser flash), allowing them to decay in an initial drift path, very rapidly lifting their ambient potential to a second acceleration potential during their flight through a small potential cell (a potential lift) and accelerating them in a second acceleration region into a second drift region. The second drift region can be at the same potential as the first drift region and both drift regions are preferably operated at the ground or chassis potential. In the second drift region, very light ions then possess the minimum energy provided by the second acceleration potential and the parent ions which have not decayed have the maximum energy corresponding to the sum of the first and second accelerations.
Such a mass spectrometer already can be used to analyze daughter ions in a linear mode (without using an ion reflector). However, it is more favorable to increase the performance of the instrument by an ion reflector.
If a reflector is able to reflect particles with energy deviations corresponding to about 30% of the maximum energy and the second acceleration potential provides about 70% of the total energy, then the reflector will be able to reflect all the daughter ions in a single voltage adjustment and the entire daughter-ion spectrum can be acquired in a single spectrum acquisition step.
The potential lift itself can be also used to select the parent ions for the daughter ion spectrum. However, it is more favorable to use an additional selector which can produce a better time resolution for the parent ions, i.e. for separating the selected parent ions from other potential ions of similar masses.
However, this very simple arrangement still has disadvantages. In the first place, the mass resolution produced by the velocity focusing function of the delayed acceleration in the ion source can only be adjusted relatively well at for one mass in the spectrum, and adjustment for all other masses is very poor. Secondly, the daughter-ion spectrum as a whole does not show particularly good mass resolution, which means that the signal-to-noise ratio is not very good either.
The invention consists of a potential lift device which is equipped with a power supply for velocity spread focusing by delayed acceleration of the ions after lifting the potential, thus making it possible to produce a focus of the velocity spreads of ions at the detector. In addition, it is possible to facilitate the adjustment of the mass spectrometer by dynamically shaping the acceleration pulse of the lift device to focus the velocity spreads of all ion masses in the spectrum on the detector. This is particularly useful for daughter-ion spectrum acquisition, providing improved mass resolution, signal-to-noise ratio and detection sensitivity for all masses in the spectrum.
The basic idea of the invention is to generate a spatial distribution of ions of the same mass which is correlated with different velocities inside the potential lift cell, and to use space-velocity correlation focussing for the ions to get better resolved daughter ion spectra. The expression xe2x80x9clift cellxe2x80x9d is used here not only for a completely closed cell, it is also used for the space between two adjacent, parallel grids, forming an essentially open cell. The focusing can be performed, for example, by lifting the two grids limiting the lift cell to two slightly different potentials. The focusing can be also performed by delaying the ion post-acceleration, with respect to the lifting event of the potential, in a subsequent post-acceleration region, in a similar manner as in the method of delayed ion acceleration (delayed with respect to the ion-generating laser flash) in the ion source. In both cases it is the aim to velocity-focus the ions by their space-velocity correlation, according to the known principle of Wiley and McLaren. More than one post-acceleration region can be connected to the potential lift so that it will not be necessary to switch the full acceleration voltage, thereby gaining an additional adjustment parameter.
To generate a correlated spatial distribution of ions of the same mass but different velocities within the potential lift cell or the adjacent acceleration region, the locus of the velocity focusing for the ions by delayed acceleration in the ion source no longer has to be positioned to fall into the potential lift cell. The delayed acceleration of ions within an ion source is well-known and need not to be described here. The delay of the acceleration is a delay with respect to the ionization event, e.g. a laser pulse.
It is particularly beneficial to arrange an ion selector between the ion source and potential lift. The velocity focusing for the parent ions from the delayed acceleration of the ion source is then adjusted to take place exactly at the location of the ion selector. A certain distance must be maintained between the ion selector and the potential lift so that the ions disperse again when entering the potential lift because they are travelling at slightly different velocities. It is the so-produced correlation between location and velocity inside the potential lift cell which allows a second velocity focusing by delayed acceleration in the lift region.
This invention can be used already in linear time-of-flight mass spectrometers. The second velocity focusing of the lift cell arrangement is then directly directed onto the ion detector.
In combination with a two-stage reflector, velocity focusing can be achieved at the detector in the same spectrum both for the parent ions and for the fragment ions of all masses produced from them, thus yielding high mass resolution over the entire daughter ion spectrum. Within limits, the focal length for velocity focusing of light ions and of heavy ions can be adjusted at will in a two-stage reflector by selecting the reflector potential and geometry.
It is, however, a complicated process to find the best adjustment of the time-of-flight mass spectrometer to achieve high resolution throughout the whole daughter ion spectrum. The best adjustment requires alteration of the distances between the ion source and the selector, the potential lift, the two-stage reflector and the detector, it requires variation of the voltages at the reflector and potential lift and variation of the delay-time for the post acceleration caused by the potential lift or its acceleration fields. Thus, the adjustment requires a large amount of experimentation. Simulation using appropriate simulation programs is also very time consuming.
For this reason, another idea of the invention is to replace the mechanical distance adjustments which are difficult to carry out, by introducing purely electronically controllable parameters. The idea consists of dynamically varying the voltages at the potential-lift acceleration regions after switching on the acceleration, i.e. applying shaped acceleration pulses, so that ions of all masses in the spectrum experience optimum velocity focusing at the detector.
The basic principle of such pulse-shaped acceleration pulses in combination with delayed acceleration and the resulting effects is already known from U.S. Pat. No. 5,969,348 (DE 196 38 577) where the dynamic delayed acceleration in the ion source is used to achieve high resolution throughout the spectrum.
Normally, delayed acceleration has the effect of giving light ions a shorter travelling distance before they are velocity focused than heavier ions. However, a distribution of focus sites for the velocities of ions of different masses such as this can only be imaged on the detector by subsequent reflection using velocity focusing if the ratios between all the distances in the mass spectrometer are geometrically favorable. Using the standard geometrical design of time-of-flight mass spectrometers, the reflector also has a shorter focal length for velocity focusing in the case of lighter ions. This type of geometry requires an intermediate velocity focus which is nearer to the reflector for light ions than it is for heavier ions, so that ions of all masses in the spectrum velocity-focus at the detector. However, the delayed acceleration in the potential lift provides a distribution of velocity-focal points where the heavier ions focus nearer to the reflector.
By dynamically changing the post-acceleration fields at the potential lift in time after the acceleration has been switched on, it is possible to reverse the distribution of intermediate focus sites so that light ions are velocity focused after a longer path, i.e. nearer to the reflector, than the one for the heavier ions. This configuration can more favorably focused by the reflector onto the detector.
It is even possible to make use of the fact that the lift potential and the post-acceleration voltages cannot be switched instantaneously on a nanosecond scale due to supply lead inductances and stray capacities. The potentials always show a time constant and creep more or less exponentially towards the final value. Targeted adaptation of these time constants and transients is in most cases sufficient to achieve the desired effect. For even better results, the time constant can also be made adjustable.
It is therefore possible to measure parent and fragment ions in the mass range from 60 to 3000 atomic mass units simultaneously with the isotopes resolved throughout the entire mass range. This mass range is of particular interest in the structural elucidation of peptides. Due to the good mass resolution, the now narrower mass signals are significantly higher, therefore displaying an improved ratio of signal height to noise. Because the narrow, high mass signals are more easily distinguished from the background noise, an improved detector sensitivity is also achieved.